CN102911373A - Preparation method of HPEI (hyperbranched polyethyleneimine)-encapsulated iron oxide magnetic nano particles - Google Patents
Preparation method of HPEI (hyperbranched polyethyleneimine)-encapsulated iron oxide magnetic nano particles Download PDFInfo
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Abstract
The invention relates to a preparation method of HPEI (hyperbranched polyethyleneimine)-encapsulated ferroferric oxide magnetic nano particles, comprising the steps of: adding Fe source to ultrapure water, then adding NH3.H2O, stirring in air, adding HPEI, and reacting at 134-140 DEG C for 3h, wherein the mass ratio of Fe source to HPEI is 1-5 :1; cooling, and carrying out washing and magnetic separation on precipitate to obtain HPEI-encapsulated ferroferric oxide nano particles Fe3O4/HPEI; and then carrying out different surface modification on the Fe3O4/HPEI nano particles, such as polyethylene glycolation (PEGlation), acetylation and carboxylation, so as to increase biocompatibility of the nano particles to be used for MRI (magnetic resonance imaging) diagnosis. The process is simple, the reaction conditions are mild and operation and separation are easy; and the prepared iron oxide magnetic nano particles have excellent biocompatibility and T2 relaxation effect, and have potential application value in the field of MRI imaging diagnosis.
Description
Technical field
The invention belongs to the preparation field of iron oxide magnetic nano particle, particularly a kind of preparation and method of modifying of iron oxide magnetic nano particle of HPEI parcel.
Background technology
In recent years, ferroferric oxide magnetic nanoparticle (Fe
3O
4) be widely used in various biomedical sectors, such as cellular segregation and purifying, the conveying of magnetic control cancer therapy drug, tumour magnetic thermotherapy and Magnetic resonance imaging (MRI).Up to now, Fe
3O
4Roughly can be divided into two kinds of synthetic methods of liquid phase method and solid phase method, its difference is that liquid phase method carries out building-up reactions in liquid phase, and the solid phase rule is not having the solution process just can generate target product.Solid phase method is divided into again ball milled and high temperature thermal decomposition method.But prepared sample products purity is not high, and size distribution is mixed and disorderly, and oxidizing reaction easily occurs.Liquid phase method has hydrothermal synthesis method, sol-gel method, chemical coprecipitation, hydrolysis method and electrochemical process Four types.The common granularity of the nanoparticle of Liquid preparation methods is little, and working method is easy, and lower production cost is arranged.And the hydrothermal method of development in recent years has been proved to be one of effective ways of synthetic high quality magnetic crystal grain.In its reaction and crystal growing process, reduced the generation of assembling under the hydrothermal condition, thereby formed the Z 250 crystal grain of monocrystalline.The nanoparticle that is synthesized has the advantages such as good dispersion, narrow diameter distribution, good crystalline and byproduct are few in solution.In addition, in reaction solvent, add tensio-active agent, can effectively separate nucleus, and in crystal growing process subsequently, intervene crystallization process, thereby dwindle particle size distribution and reduce particle diameter.Exposed ferroferric oxide nano granules is because high chemical reactivity is very easy to oxidation, large specific surface area is easily assembled nano particle and is caused magnetic and dispersiveness to reduce simultaneously, in addition, with human body in the suitable environment of pH value, nano level iron oxide particle is also easily assembled, and has limited its popularization and application.Up to now, solution is: process nano grain surface with physics, chemical process, usually coat the inorganic or organism of one deck at particle surface, promote its stability, water dispersible and biocompatibility.Present common method of modifying also has surface chemical modification method, sol-gel modification method, precipitin reaction modification method, polymer overmold modification method, self-assembled modified method etc.
Synthesized the iron oxide magnetic nano material that a kind of APTS modifies with a step hydrothermal synthesis method, the method technique is simple before, and reaction conditions is gentle, and easy handling separates; The iron oxide magnetic nano particle of preparation can be dispersed in the group water solution for a long time, does not have agglomeration to occur, and (history on the sunny side for Shen Mingwu, Cai Hongdong to have potential using value in the MRI image-forming diagnose.The preparation method of the iron oxide magnetic nano particle that a kind of APTS modifies.Chinese invention patent, application number: 201110104443.1, the date of application: 2011/4/26).But the iron oxide magnetic nano scantling too little (6.5nm) that the APTS of preparation modifies, particle diameter are difficult to control.
And hyperbranched polyethyleneimine (Hyperbranched Polyethyleneimine, HPEI) not only can improve the aqueous solution dispersiveness of nano particle, and numerous surface amino groups is arranged, and can be used for the further functionalized modification of nano-material surface.Shen etc. modify carboxylated multi-walled carbon nano-tubes with HPEI; the aqueous solution that has improved carbon nanotube is dispersed; simultaneously again by acetylize and carboxylated; to change its surface charge performance; obtain the neutral carbon nano-tube material with carrying negative charge of surface electrical; and studied its vitro cytotoxicity; the result shows the carbon nanotube that HPEI modifies because amino effect demonstrates cytotoxicity; through acetylize and carboxylated after derivative then do not have obvious cytotoxicity (Shen, J.Phys.Chem.C 2009.MWCNTs).
PEG (polyoxyethylene glycol) is the good polymkeric substance of a kind of physiologically acceptable, can reduce the absorption of protein and the picked-up of scavenger cell as coating material decorated nanometer particle surface, thereby prolong the nano particle transformation period in vivo, PEG has been widely used in biomedical sector.The introducing of PEG can strengthen the wetting ability of nano particle and water-soluble, promotes its biocompatibility and prolongs blood circulation time.Peng etc. utilize PEG to modify dendrimer, as template, preparation dendrimer parcel gold nano grain is used for the CT imaging, effectively improved the upper carrying capacity of gold, and improved the biocompatibility of material, can be used for preferably CT imaging (Peng, Biomaterials in the mice with tumor body, 2011, [(Au
0)
n-G5.NHAc-mPEG
20] DENPs).
Retrieval is the Fe of relevant HPEI protection both at home and abroad
3O
4Synthetic document and the patent results of nano particle shows, with the Fe of the synthetic HPEI protection of a step hydrothermal synthesis method
3O
4Nanoparticle and the research that its surface is modified effectively with modification yet there are no relevant report.
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method of iron oxide magnetic nano particle of HPEI parcel, this method technique is simple, reaction conditions is gentle, easy handling separates, the iron oxide magnetic nano particle of preparation can be dispersed in the aqueous solution for a long time, does not have agglomeration to occur; Used modifier HPEI is cheapness and environment-friendly material, has the prospect of industrialized implementation; The Fe that this method forms
3O
4/ HPEI surface is further functionalization and modification also, improves its stability and biocompatibility, and potential using value is arranged in the MRI image-forming diagnose.
The preparation method of the iron oxide magnetic nano particle of a kind of HPEI parcel of the present invention comprises:
(1) the Fe source is added in the ultrapure water, add again NH
3H
2O also stirred in air, adds the hyperbranched polyethyleneimine HPEI aqueous solution of 0.02-0.12g/mL again, in 134 ~ 140 ℃ of reactions 3 hours; Reaction naturally cools to room temperature after finishing, and after washing of precipitate magnetic is separated, namely gets the ferroferric oxide nano granules Fe of HPEI parcel
3O
4/ HPEI;
(2) Fe
3O
4/ HPEI and methoxy poly (ethylene glycol) carboxylic acid (mPEG-COOH) carry out the PEGization reaction: with methoxy poly (ethylene glycol) carboxylic acid (mPEG-COOH) and 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) is dissolved in respectively among the 3-6mL DMSO, behind the stir-activating 3h, add again the Fe of step (1) preparation
3O
4/ HPEI vibrated 3 days, used the hydromagnetic separating, washing again, among redispersion entry or the PBS, obtained the Fe of PEGization
3O
4/ HPEI.PEG nano particle;
Or Fe
3O
4/ HPEI and diacetyl oxide carry out acetylization reaction: with the Fe of step (1) preparation
3O
4/ HPEI nano particle is dispersed in the water, after triethylamine fully mixes, dropwise adds diacetyl oxide.Stirring reaction 24 ~ 48 hours is used the hydromagnetic separating, washing, makes acetylizad Fe
3O
4/ HPEI.Ac nano particle;
Or Fe
3O
4/ HPEI and butanedioic anhydride carry out carboxylation reaction: with the Fe of step (1) preparation
3O
4/ HPEI nano particle is dispersed among the DMSO, dropwise adds the DMSO solution that contains succinyl oxide, and stirring reaction 24 ~ 48 hours is used the hydromagnetic separating, washing, makes the Fe that carboxylated surface is negative charge
3O
4/ HPEI.SAH nano particle.
Fe source in the described step (1) is FeCl
24H
2O.
Fe source, ultrapure water, NH in the described step (1)
3H
2The proportioning of O is 1g:6-51mL:5mL.
Source of iron in the described step (1) and hyperbranched polyethyleneimine HPEI mass ratio are 1-5:1.
NH in the described step (1)
3H
2The O mass percent concentration is 25-28%.
The molecular weight Mw=25000 of hyperbranched polyethyleneimine HPEI in the described step (1).
The molecular weight of the methoxy poly (ethylene glycol) carboxylic acid (mPEG-COOH) in the described step (2) is 2000.
Methoxy poly (ethylene glycol) carboxylic acid (mPEG-COOH) in the described step (2) is 1:5-10 with the mol ratio that is wrapped in the amino on ferroferric oxide magnetic nanoparticle surface.
Diacetyl oxide in the described step (2) or succinyl oxide and Fe
3O
4Among/the PEI-NH
2Mol ratio be 5-10:1.
Triethylamine, diacetyl oxide and Fe in the described step (2)
3O
4Among/the PEI-NH
2Mol ratio is 5:5:1.
The present invention utilizes polymine to carry out the ferroferric oxide magnetic nanoparticle of the controlled HPEI parcel of the synthetic size of one step hydro thermal method, and can further modify and functionalization its surface.Utilize hyperbranched polymer hyperbranched polyethyleneimine HPEI to modify and stabilization iron oxide magnetic material, be expected to the size of the feed ratio control synthesis of nano ferric oxide particles by HPEI and molysite, utilize simultaneously the amino of polymine HPEI can adorned advantage can be to the further modification in ferric oxide nanometer particle surface.
The present invention studies different parameters to Fe
3O
4The impact of material surface modification, size, shape, structure and stability is such as water and the ammoniacal liquor of different volumes, the FeCl of different mass ratio
24H
2O and hyperbranched polyethyleneimine are at first with FeCl
24H
2O in air after the oxidation, under high pressure in 134 ℃ and in the presence of HPEI, the ferroferric oxide nano granules of Hydrothermal Synthesis HPEI protection.The Fe of synthetic HPEI protection
3O
4Nanoparticle size is 11-22nm, and the amino with the peripheral HPEI of nano particle carries out PEGization, acetylize and carboxylated subsequently, and to improve its biocompatibility, the ferric oxide nanometer particle of the method preparation can potentially be diagnosed for the MRI molecular image.
The present invention uses the methods such as transmission electron microscope (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), inductively coupled plasma atomic emission spectrometry (ICP-AES) and Zeta electric potential and scattering of light kinetics to characterize the magnetic nanoparticle for preparing, nano particle T
2Relaxivity records by magnetic resonance imager, utilizes simultaneously stability test, blood compatibility, mtt assay to check the stability of nano particle, blood compatibility and cytotoxicity, and concrete test result is as follows:
(1) test result of X-ray diffraction (XRD)
By comparison and the analysis to diffracting spectrum, the material that is synthesized and Fe
3O
4Collection of illustrative plates (ICSD 20-596) consistent, show the Fe that HPEI modifies
3O
4Crystalline structure does not change, and product is Z 250.In addition, according to the Debye-Scherrer formula (D=k λ/β cos θ, λ=0.15406nm), get maximum peak place (311) and calculate:
Fe
3O
4/HPEI(1):11.5nm；
Fe
3O
4/HPEI(2):17.5nm；
Fe
3O
4/HPEI(3):18.3nm；
Fe
3O
4/HPEI(4):18.9nm。
With reference to the accompanying drawings 1 and table 1;
(2) thermogravimetric analysis (TGA) test result
In order to compare four kinds of Fe that the different ratios of raw materials single stage method is synthesized
3O
4The modification amount of nano grain surface HPEI, the Fe synthetic to hydrothermal method
3O
4The Fe synthetic with single stage method
3O
4/ HPEI has carried out the TGA test.As seen from Figure 2, with pure iron oxide relatively, in No. 1 sample HPEI to upload rate the highest, be in 9.69%, No. 3 sample HPEI to upload rate minimum, be 7.34%, as can be seen from Figure 3, No. 1 and No. 3 samples carry out the thermogravimetric test after by HPEI surface amino groups and PEG molar ratio 5:1 reaction; No. 1 Fe
3O
4/ HPEI.PEG(Fig. 3 a) weightlessness is 14.87%; No. 3 Fe
3O
4/ HPEI.PEG(Fig. 3 b) weightlessness is 10.70%.Therefore can calculate No. 1 Fe
3O
4/ HPEI.PEG and No. 3 Fe
3O
4The rate of uploading of the PEG of/HPEI.PEG is respectively 5.18% and 3.36%, shows that thus 1 and No. 3 sample successfully modified up by PEG.As seen, the nano grain surface of being modified by the synthetic HPEI of this law contains the more amino of crypto set, can require further to carry out functionalization or finishing according to different biomedical applications.The TGA test result shows that HPEI and PEG successfully are modified at Fe
3O
4On the nano grain surface.With reference to the accompanying drawings 2 and 3;
(3) test result of Fourier transform infrared spectroscopy (FTIR)
Fe
3O
4/ HPEI and pure Fe
3O
4The FTIR spectrogram as shown in Figure 4.For Fe
3O
4/ HPEI nano particle, 2930 and 2850cm
-1Be the stretching vibration of C-H, 1126cm
-1Be the stretching vibration of C-N key.In addition, 3450 and 1630cm
-1The absorption peak at place is then owing to-NH
2With the residual water in the sample.With pure Fe
3O
4Nano particle is compared, because-NH
2Existence, Fe
3O
4/ HPEI is 3450 and 1630cm
-1Place's absorption peak area is larger.FTIR result shows that HPEI successfully is modified at Fe
3O
4On the nano grain surface.With reference to the accompanying drawings 4;
(4) transmission electron microscope (TEM) test result
The TEM test result shows Fe
3O
4The pattern of/HPEI is sphere or torispherical, by to Fe
3O
4/ HPEI nano particle respectively the diameter of random measurement 200-300 the nano particle mean diameter that calculates No. 1 sample is 11.50Hnm; No. 2 sample average diameters are 20.2nm; No. 3 sample average diameters are 16.7nm; No. 4 sample average diameters are 21.8nm.The result shows that the size of No. 1 sample is less, and the mean diameter of 2-4 sample is relatively large, and the HPEI of different amounts is little on the size impact of material, but FeCl
2`4H
2The consumption of O has certain impact to structure and the size of particle, relative No. 1 sample, and 2-4 number the more regular homogeneous of structure crystal formation, size is larger.These size results are consistent with the XRD test result.Can find out among the TEM figure that particle is with one deck macromole shell outward and surrounds, show that HPEI is modified at Fe equably
3O
4Nano grain surface.With reference to the accompanying drawings 5 and table 1;
1H is with respect to 1 and No. 3 sample of unmodified PEG, because the modification of the PEG of particle periphery causes Fe
3O
4/ HPEI.PEG size increases slightly, and can find out that the macromole layer of particle periphery thickens by naked eyes from TEM figure, in conjunction with TG(Fig. 2,3) result, proved that all PEG successfully modifies particle surface.With reference to the accompanying drawings 6 and table 2;
(5) nano particle Zeta electric potential and light scattering test result
The peripheral amino of nano particle can produce cytotoxicity and with the cytolemma non-specific binding, thereby limited the biologic applications of these nano particles.We find in research in the past, and neutralization is modified at the amino of the polymine of multi-wall carbon nano-tube tube-surface, toxicity (Shen, J.Phys.Chem.C 2009.MWCNTs, 113 (8): 3150-3156) that can effectively reduce multi-walled carbon nano-tubes.Equally, we pass through Fe
3O
4The peripheral amino of/HPEI carries out PEGization, acetylize and carboxylated, makes its surface potential be neutral or electronegative, improves the biocompatibility of nano particle.
Potential determination result (table 3) shows, because the peripheral a large amount of amino, No. 1 Fe of existing
3O
4The surface potential of/HPEI is about 38mV.Through after the acetylization reaction; potential drop is to being about 26mV; electromotive force has obvious decline; but also not reaching electric neutrality, may be because the HPEI part is amino and ferric oxide interacts, so that their reactive behavior weakens; fully acetylize; so also present a part of positive polarity, but because they and ferric oxide effect, so can't be as simple HPEI to the toxicity of cell.And the particle surface electromotive force is negative electricity behind the carboxylation reaction, No. 1 Fe
3O
4/ HPEI electromotive force is reduced to-17.3mV from 38.07mV, and the result shows, the carboxylation reaction success, and amino fully reacts.And through after the PEGization reaction, Fe
3O
4The electromotive force of/HPEI.PEG also descends.These presentation of results the successful implementation of PEGization, acetylize and carboxylation reaction.With reference to subordinate list 3;
(6) stability test
Stability test research surface is through different modifying and with the stability of different charge material in different media.At first respectively with No. 1 Fe
3O
4/ HPEI, Fe
3O
4/ HPEI.Ac, Fe
3O
4/ HPEI.SAH and Fe
3O
4Four kinds of materials of/HPEI.PEG, be positioned in pure water, PBS damping fluid (pH 7.4) and the substratum place a week after, Fe
3O
4/ HPEI, Fe
3O
4/ HPEI.Ac, Fe
3O
4/ HPEI.PEG stably dispersing, precipitation and gathering do not occur, and Fe is described
3O
4/ HPEI, after the acetylize and the derivative after the PEGization in solution, have good dispersiveness and stable.And certain clustering phenomena appears in the electronegative carboxylated material in surface, but still is dispersed at an easy rate in the aqueous solution.
(7) T
2The relaxation rate measuring result
Fe
3O
4Nano material is a kind of MRI negative contrast medium, can reduce strength of signal.Relaxation rate (r
2) reflected and the efficient of nanoparticle as the MRI contrast medium be the T2 of every mmole iron, can pass through relaxation time (T
2) inverse calculate.Fig. 7 a and b are respectively No. 1 and No. 3 Fe
3O
4The corresponding Linear Fit Chart of/HPEI material can be found out Fe
3O
4The relaxation rate of/HPEI material is along with the increase (in the 0-0.02mM concentration range) of concentration of iron has good linearly dependent coefficient.From linear relationship chart as can be known, Fe
3O
4Modify upper PEG even/HPEI material is peripheral, also do not stopped itself and the contact rate of proton, on not too large impact of relaxation rate, and all have good relaxation rate (130-160mM
-1s
-1).Therefore, the Fe that the present invention synthesized
3O
4/ HPEI and Fe
3O
4/ HPEI.PEG nano material can be used as good T
2The signal attenuation contrast medium is expected to be applied to the diagnosis of MRI molecular imaging; With reference to the accompanying drawings 7.
(8) blood compatibility
Can be better be used for bio-imaging in the body for the ease of material, this test determination No. 1 Fe
3O
4/ HPEI material through different modifying with the blood compatibility behind the different electric charges.Shown Fe among Fig. 8 No. 1
3O
4/ HPEI(Fig. 8 a), Fe
3O
4/ HPEI.PEG(Fig. 8 b), Fe
3O
4/ HPEI.Ac(Fig. 8 c) and Fe
3O
4/ HPEI.SAH(Fig. 8 d) the hemolytic test result under different concns 50,100,200,400 μ g/mL.By the absorbancy measurement of supernatant liquid being come the hemolytic of quantitative evaluation sample.Show such as Fig. 8 upper right corner uv-spectrogram, reaching under the 400 μ g/mL conditions Fe
3O
4/ HPEI, Fe
3O
4/ HPEI.PEG and Fe
3O
4The percentage of hemolysis of/HPEI.Ac is all less than 5%, but Fe
3O
4The hemolysis rate of/HPEI.SAH is 7.65%.And when being lower than 200 μ g/mL, the hemolysis rate of all material is all very low, thereby can make them be successfully used to biological in-vivo imaging; With reference to the accompanying drawings 8.
(9) MTT cell viability test result
Vigor by MTT colorimetric method for determining KB cell (a kind of cell strain of human epithelium's cancer) detects institute's synthetic materials and surperficial through the cytotoxicity after the difference in functionality modification.The KB cell respectively with Fe
3O
4/ HPEI, Fe
3O
4/ HPEI.PEG, Fe
3O
4/ HPEI.Ac and Fe
3O
4/ HPEI.SAH cultivated 24 hours under concentration is 10,25,50 and 100 μ g/mL and 37 ℃ altogether.Then, after processing, MTT measures light absorption value at 570nm place, and according to propagation and the vigor of this value calculating cell.The statistical of the impact of material on cell proliferation and control group under the different concns (damping fluid PBS, pH 7.4) is then implemented by the one-way analysis of variance method.Compare with control group, the surface is through the Fe of modification and functionalization
3O
4/ HPEI.PEG, Fe
3O
4/ HPEI.Ac and Fe
3O
4/ HPEI.SAH in experimental concentration 0 to 100 μ g/mL scope to the survival rate of KB cell showing property difference not.And Fe
3O
4/ HPEI begins to occur cytotoxicity (p＜0.05) when dosage reaches 50 μ g/mL, this shows it is unfavorable for cell when high density propagation, may be that toxic and surperficial high positive charge cell growth has certain influence to cell owing to surperficial HPEI.And the surface is through the Fe of modification and functionalization
3O
4/ HPEI.PEG, Fe
3O
4/ HPEI.Ac and Fe
3O
4/ HPEI.SAH then has good biocompatibility, is convenient to various biomedical applications; With reference to the accompanying drawings 9.
Beneficial effect
(1) " step " hydrothermal method that the present invention adopts simply, HPEI is auxiliary is come the Fe of the good HPEI protection of synthesizing water-solubility
3O
4Nanoparticle, this method also can synthesize by the control different ratios of raw materials iron oxide magnetic nano particle of different size, and technique is simple, reaction conditions is gentle, easy handling separates, and used modifier is cheap and eco-friendly polymine material, has the prospect of industrialized implementation;
(2) the iron oxide magnetic nano particle of the present invention's preparation can be dispersed in the group water solution for a long time, do not have agglomeration to occur, and PEGization, acetylize and carboxylated iron oxide magnetic nano particle has good biocompatibility and higher T
2Relaxation rate can be applied to the MRI image-forming diagnose potentially.
Description of drawings
Fig. 1 is the Fe that the present invention prepares
3O
4The X-ray diffractogram of/HPEI; Fe
3O
4/ HPEI (1-4) is respectively the 1-4 sample;
Fig. 2 is the Fe that the present invention prepares
3O
4And Fe
3O
4The thermogravimetric analysis figure of/HPEI; Fe
3O
4/ HPEI (1-4) is respectively the 1-4 sample; Naked Fe
3O
4Unprotected pure Fe for Comparative Examples 1 preparation
3O
4
Fig. 3 is 1 and No. 3 Fe that the present invention prepares
3O
4/ HPEI is through Fe after the PEGization
3O
4The thermogravimetric analysis figure of/HPEI.PEG; Fe
3O
4Unprotected pure Fe for Comparative Examples 1 preparation
3O
4
Fig. 4 is the Fe that the present invention prepares
3O
4And Fe
3O
4The Fourier transform infrared spectroscopy figure of/HPEI, a, b, c, d and e are respectively No. 1, and No. 2, No. 3, No. 4 Fe
3O
4The infrared spectrum of/HPEI and unprotected pure Fe
3O
4Infrared spectrum;
Fig. 5 is 1 (a) that the present invention prepares, 2 (b), 3 (c) and 4 (d) number Fe
3O
4The transmission electron microscope picture of/HPEI and size distribution plot;
Fig. 6 is that 1 (a) and 3 (b) number sample that the present invention prepares obtains Fe through PEGization
3O
4The transmission electron microscope picture of/HPEI.PEG and size distribution plot;
Fig. 7 is 1 (a) and 3 (b) number Fe that the present invention prepares
3O
4/ HPEI and Fe
3O
4The linear relationship chart of the relaxation rate of/HPEI.PEG;
Fig. 8 is No. 1 Fe that the present invention prepares
3O
4/ HPEI(a), Fe
3O
4/ HPEI.PEG(b), Fe
3O
4/ HPEI.Ac(c) and Fe
3O
4/ HPEI.SAH(d) the hemolytic experiment uv-spectrogram of (concentration range 50-400 μ g/mL);
Fig. 9 is KB cell process PBS damping fluid (contrast), the Fe of mtt assay test
3O
4/ HPEI, Fe
3O
4/ HPEI.PEG, Fe
3O
4/ HPEI.Ac and Fe
3O
4/ HPEI.SAH nano particle (concentration range is at 0-100 μ g/mL) is processed the cell viability after 24 hours.
Figure 10 is the molecular structural formula of hyperbranched polyethyleneimine HPEI.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
With 1.2519g FeCl
24H
2O adds in the beaker, adds the ultrapure water (resistivity is greater than 18.2M Ω .cm) of 7.75mL, in constantly stirring, adds 6.25mL NH
3H
2O with above-mentioned mixed solution continuously stirring 10 minutes in air, thereby guarantees that ferrous iron is fully oxidized, then with reactant transfer in reactor.0.53879g HPEI is dissolved in the 5mL aqueous solution, behind the ultrasonic dissolution, with liquid-transfering gun it is changed in the reactor, with the abundant mixing of solution in the reactor, in 134 ~ 140 ℃ of reactions approximately 3 hours; Reaction naturally cools to room temperature, with resulting black precipitate Fe after finishing
3O
4/ HPEI is scattered in the ultrapure water, magnetic separates, and again dispersion, magnetic separate, and so repeats five pure water washings, to remove excessive reaction reagent, again is scattered in aqueous phase, namely gets ferroferric oxide nano granules (No. 1 Fe of polymine protection
3O
4/ HPEI); Reference table 1;
Characterize No. 1 Fe by XRD, TG, FTIR, TEM, Zeta testing method
3O
4/ HPEI material, XRD result has shown Fe No. 1
3O
4The crystalline structure of/HPEI nano particle does not change, and product is Z 250.In addition, according to Debye-Scherrer formula (D=k λ/β cos θ, λ=0.15406nm), get maximum peak place (311) and calculate Fe No. 1
3O
4/ HPEI size 11.5nm(reference table 1 and accompanying drawing 1); With pure iron oxide relatively, No. 1 Fe
3O
4The rate of uploading of HPEI is 9.69% among the/HPEI, shows thus Fe No. 1
3O
4The HPEI of/HPEI successfully modifies up (with reference to the accompanying drawings 2).And further by No. 1 Fe of FTIR spectral characterization
3O
4/ HPEI and pure Fe
3O
4Sample is for No. 1 Fe
3O
4/ HPEI nano particle, 2930 and 2850cm
-1Be the stretching vibration of C-H, 1126cm
-1Be the stretching vibration of C-N key.In addition, 3450 and 1630cm
-1The absorption peak at place is then owing to-NH
2With the residual water in the sample.With pure Fe
3O
4Nano particle is compared, because-NH
2Existence, No. 1 Fe
3O
4/ HPEI is 3450 and 1630cm
-1Place's absorption peak area is larger, shows that qualitatively HPEI successfully modifies up (with reference to the accompanying drawings 4a and 4e).TEM result shows Fe
3O
4The pattern of/HPEI is sphere or torispherical, by to No. 1 Fe
3O
4The diameter of 200-300 nano particle of/HPEI nano particle random measurement calculates Fe No. 1
3O
4The mean diameter of/HPEI is 11.53Hnm, and size results is consistent with XRD result, and ferric oxide is with a layer macromole transparency material parcel outward and shows further that also HPEI successfully is modified at iron oxide surface (with reference to the accompanying drawings 5a and table 1).The Zeta test result shows No. 1 Fe
3O
4/ HPEI positively charged (38.07mV), ydrodynamics diameter are about 310.4nm(with reference to subordinate list 3).These results show that HPEI is modified at Fe equably
3O
4On the nano grain surface.
(1) with 0.252g FeCl
24H
2O adds in the beaker, adds in the ultrapure water (resistivity is greater than 18.2M Ω .cm) of 12.75mL again, in constantly stirring, adds 1.25mL NH
3H
2O with above-mentioned mixed solution continuously stirring 10 minutes in air, thereby guarantees that ferrous iron is fully oxidized, then with reactant transfer in reactor.0.05153g HPEI is dissolved in the 5mL aqueous solution, behind the ultrasonic dissolution, with liquid-transfering gun it is changed in the reactor, with the abundant mixing of solution in the reactor, in 134 ~ 140 ℃ of reactions approximately 3 hours; Reference table 1; Reaction naturally cools to room temperature, with resulting black precipitate Fe after finishing
3O
4/ HPEI is scattered in the ultrapure water, magnetic separates, and again dispersion, magnetic separate, and so repeats five pure water washings, to remove excessive reaction reagent, again is scattered in aqueous phase.Namely get No. 2 Fe of ferroferric oxide nano granules of polymine parcel
3O
4/ HPEI; With reference to subordinate list 1;
Characterize No. 2 Fe of material by XRD, TG, FTIR, TEM testing method
3O
4/ HPEI, XRD result have shown No. 2 Fe that HPEI modifies
3O
4The crystalline structure of/HPEI nano particle does not change, and product is Z 250.In addition, according to Debye-Scherrer formula (D=k λ/β cos θ, λ=0.15406nm), get maximum peak place (311) and calculate Fe No. 2
3O
4/ HPEI size 17.5nm(reference table 1 and accompanying drawing 1); With pure iron oxide relatively, No. 2 Fe
3O
4The rate of uploading of HPEI is 8.13% among the/HPEI, shows thus Fe No. 2
3O
4The HPEI of/HPEI successfully modifies up (with reference to the accompanying drawings 2).And further by No. 2 Fe of FTIR spectral characterization
3O
4/ HPEI and pure Fe
3O
4Sample is for No. 2 Fe
3O
4/ HPEI nano particle, 2930 and 2850cm
-1Be the stretching vibration of C-H, 1126cm
-1Be the stretching vibration of C-N key.In addition, 3450 and 1630cm
-1The absorption peak at place is then owing to-NH
2With the residual water in the sample.With pure Fe
3O
4Nano particle is compared, because-NH
2Existence, No. 2 Fe
3O
4/ HPEI is 3450 and 1630cm
-1Place's absorption peak area is larger, shows that qualitatively HPEI successfully modifies up (with reference to the accompanying drawings 4b and 4e).TEM result shows Fe
3O
4The pattern of/HPEI is sphere or torispherical, peripheral passing through No. 2 Fe
3O
4The diameter of 200-300 nano particle of/HPEI nano particle random measurement calculates Fe No. 2
3O
4The mean diameter of/HPEI is 20.24Hnm, and ferric oxide is with a layer macromole transparency material parcel outward and shows further that also HPEI successfully is wrapped in iron oxide surface (with reference to the accompanying drawings 5b and table 1).These results show that HPEI is modified at Fe equably
3O
4On the nano grain surface.
(1) with 0.25294g FeCl
24H
2O adds in the beaker, in the ultrapure water (resistivity is greater than 18.2M Ω .cm) that adds 12.75mL, in constantly stirring, adds 1.25mLNH
3H
2O with above-mentioned mixed solution continuously stirring 10 minutes in air, thereby guarantees that ferrous iron is fully oxidized, then with reactant transfer in reactor.0.1037g HPEI is dissolved in the 5mL aqueous solution, behind the ultrasonic dissolution, with liquid-transfering gun it is changed in the reactor, with the abundant mixing of solution in the reactor, in 134 ~ 140 ℃ of reactions approximately 3 hours; Reference table 1;
(2) after reaction finishes, naturally cool to room temperature, with resulting black precipitate Fe
3O
4/ HPEI is scattered in the ultrapure water, magnetic separates, and again dispersion, magnetic separate, and so repeats five pure water washings, to remove excessive reaction reagent, again is scattered in aqueous phase.Namely get No. 3 Fe of ferroferric oxide nano granules of polymine parcel
3O
4/ HPEI; With reference to subordinate list 1;
Characterize No. 3 Fe of material by testing method such as XRD, TG, FTIR, TEM, Zeta electric potentials
3O
4/ HPEI, XRD result have shown No. 3 Fe that HPEI modifies
3O
4The crystalline structure of/HPEI nano particle does not change, and product is Z 250.In addition, according to Debye-Scherrer formula (D=k λ/β cos θ, λ=0.15406nm), get maximum peak place (311) and calculate Fe No. 3
3O
4/ HPEI size 18.3nm(reference table 1 and accompanying drawing 1); With pure iron oxide relatively, No. 3 Fe
3O
4The rate of uploading of HPEI is 7.34% among the/HPEI, shows thus Fe No. 3
3O
4The HPEI of/HPEI successfully modifies up (with reference to the accompanying drawings 2).And further by No. 3 Fe of FTIR spectral characterization
3O
4/ HPEI and pure Fe
3O
4Sample is for No. 3 Fe
3O
4/ HPEI nano particle, 2930 and 2850cm
-1Be the stretching vibration of C-H, 1126cm
-1Be the stretching vibration of C-N key.In addition, 3450 and 1630cm
-1The absorption peak at place is then owing to-NH
2With the residual water in the sample.With pure Fe
3O
4Nano particle is compared, because-NH
2Existence, No. 3 Fe
3O
4/ HPEI is 3450 and 1630cm
-1Place's absorption peak area is larger, shows that qualitatively HPEI successfully modifies up (with reference to the accompanying drawings 4c and 4e).TEM result shows Fe
3O
4The pattern of/HPEI is sphere or torispherical, peripheral passing through No. 3 Fe
3O
4The diameter of 200-300 nano particle of/HPEI nano particle random measurement calculates Fe No. 3
3O
4The mean diameter of/HPEI is 16.75Hnm, and size results is consistent with XRD result, and ferric oxide is with a layer macromole transparency material parcel outward and shows further that also HPEI successfully is wrapped in iron oxide surface (with reference to the accompanying drawings 5c and table 1).The Zeta test result shows No. 3 Fe
3O
4/ HPEI positively charged (39.2mV), the ydrodynamics diameter is about 116.3nm.These results show that HPEI is modified at Fe equably
3O
4On the nano grain surface.
(1) with 0.25278g FeCl
24H
2O adds in the beaker, in the ultrapure water (resistivity is greater than 18.2M Ω .cm) that adds 12.75mL, in constantly stirring, adds 1.25mLNH
3H
2O with above-mentioned mixed solution continuously stirring 10 minutes in air, thereby guarantees that ferrous iron is fully oxidized, then with reactant transfer in reactor.0.21512g HPEI is dissolved in the 5mL aqueous solution, behind the ultrasonic dissolution, with liquid-transfering gun it is changed in the reactor, with the abundant mixing of solution in the reactor, in 134 ~ 140 ℃ of reactions approximately 3 hours; Reference table 1;
(2) after reaction finishes, naturally cool to room temperature, with resulting black precipitate Fe
3O
4/ HPEI is scattered in the ultrapure water, magnetic separates, and again dispersion, magnetic separate, and so repeats five pure water washings, to remove excessive reaction reagent, again is scattered in aqueous phase.Namely get No. 4 Fe of ferroferric oxide nano granules of polymine parcel
3O
4/ HPEI; Reference table 1;
Characterize No. 4 Fe of material by testing method such as XRD, TG, FTIR, TEM
3O
4/ HPEI, XRD result have shown No. 4 Fe that HPEI modifies
3O
4The crystalline structure of/HPEI nano particle does not change, and product is Z 250.In addition, according to Debye-Scherrer formula (D=k λ/β cos θ, λ=0.15406nm), get maximum peak place (311) and calculate Fe No. 4
3O
4/ HPEI size 18.9nm(reference table 1 and accompanying drawing 1); With pure iron oxide relatively, No. 4 Fe
3O
4The rate of uploading of HPEI is 8.31% among the/HPEI, shows thus Fe No. 4
3O
4The HPEI of/HPEI successfully modifies up (with reference to the accompanying drawings 2).And further by No. 4 Fe of FTIR spectral characterization
3O
4/ HPEI and pure Fe
3O
4Sample is for No. 4 Fe
3O
4/ HPEI nano particle, 2930 and 2850cm
-1Be the stretching vibration of C-H, 1126cm
-1Be the stretching vibration of C-N key.In addition, 3450 and 1630cm
-1The absorption peak at place is then owing to-NH
2With the residual water in the sample.With pure Fe
3O
4Nano particle is compared, because-NH
2Existence, No. 4 Fe
3O
4/ HPEI is 3450 and 1630cm
-1Place's absorption peak area is larger, shows that qualitatively HPEI successfully modifies up (with reference to the accompanying drawings 4d and 4e).TEM result shows Fe No. 4
3O
4The pattern of/HPEI is sphere or torispherical, with 1-3 Fe
3O
4/ HPEI compares, No. 4 Fe
3O
4/ HPEI crystalline structure is more regular, homogeneous.And pass through No. 4 Fe
3O
4The diameter of 200-300 nano particle of/HPEI nano particle random measurement calculates Fe No. 4
3O
4The mean diameter of/HPEI is with reference to the accompanying drawings 5d and table 1 of 21.86Hnm().These results show that HPEI is modified at Fe equably
3O
4On the nano grain surface.
The Fe of the synthetic different size of table 1 differing materials proportioning
3O
4/ HPEI
Annotate: a is TEM result, and b is XRD result.
Get Fe No. 1
3O
4/ HPEI carries out the PEGization reaction, presses Fe
3O
4The molar ratio of/HPEI surface amino groups and mPEG-COOH (5:1) carries out the PEGization reaction, and such as table 2, the mPEG-COOH and the 23.00mg EDC that at first take by weighing 22.03mg are dissolved in respectively among the 3-6mL DMSO, behind the stir-activating 3h, are added to No. 1 Fe of above-mentioned preparation again
3O
4/ HPEI 2mL the aqueous solution (24.44mg), shaking table reaction 3 days is used the hydromagnetic separating, washing 5 times again, among redispersion entry or the PBS, obtains Fe No. 1
3O
4/ HPEI.PEG.
Characterize No. 1 Fe of material by testing method such as TG, TEM, Zeta electric potentials
3O
4/ HPEI.PEG, result show Fe No. 1
3O
4The weightlessness of/HPEI.PEG is 14.87%, with No. 1 Fe
3O
4/ HPEI(weightlessness is 9.69%) relatively, the rate of uploading of PEG is 5.18%, shows thus Fe No. 1
3O
4The PEG of/HPEI.PEG successfully modifies up (with reference to the accompanying drawings 3).TEM result shows Fe
3O
4The pattern of/HPEI.PEG is sphere or torispherical, by to No. 1 Fe
3O
4The diameter of 200-300 nano particle of/HPEI.PEG nano particle random measurement calculates Fe No. 1
3O
4The mean diameter of/HPEI.PEG is 127Hnm.No. 1 Fe with unmodified PEG
3O
4/ HPEI compares, because the modification of the PEG of particle periphery causes Fe
3O
4/ HPEI.PEG size increases slightly, and can find out that the macromole layer of particle periphery thickens by naked eyes from TEM figure, has therefore proved that further PEG successfully modifies particle surface (with reference to the accompanying drawings 6a and table 2); The Zeta test result shows, after the PEGization reaction, shows No. 1 Fe
3O
4/ HPEI.PEG is positively charged (30.9mV) still, with Fe
3O
4/ HPEI compares, and electromotive force descends to some extent, and the ydrodynamics diameter is about 161.9nm, dispersion of materials more stable (with reference to subordinate list 3).These results show that PEG is modified at Fe equably
3O
4On the nano grain surface.
Embodiment 6
Get Fe No. 3
3O
4/ HPEI carries out the PEGization reaction, presses Fe
3O
4The molar ratio of/HPEI surface amino groups and mPEG-COOH (5:1) carries out the PEGization reaction, and such as table 2, the mPEG-COOH and the 6.82mg EDC that at first take by weighing 4.124mg are dissolved in respectively among the 3-6mL DMSO, behind the stir-activating 3h, add No. 3 Fe of above-mentioned preparation again
3O
4/ HPEI 2mL the aqueous solution (6.04mg), shaking table reaction 3 days is used the hydromagnetic separating, washing 5 times again, among redispersion entry or the PBS, obtains Fe No. 3
3O
4/ HPEI.PEG.
Characterize No. 3 Fe of material by testing method such as TG, TEM, Zeta electric potentials
3O
4/ HPEI.PEG, result show Fe No. 3
3O
4The weightlessness of/HPEI.PEG is 10.70%, with No. 3 Fe
3O
4/ HPEI(weightlessness is 7.34%) relatively, the rate of uploading of PEG is 3.36%, shows thus Fe No. 3
3O
4The PEG of/HPEI.PEG successfully modifies up (with reference to the accompanying drawings 3).TEM result shows Fe
3O
4The pattern of/HPEI.PEG is sphere or torispherical, by to No. 3 Fe
3O
4The diameter of 200-300 nano particle of/HPEI.PEG nano particle random measurement calculates Fe No. 3
3O
4The mean diameter of/HPEI.PEG is 18.79Hnm.No. 3 Fe of 10H and unmodified PEG
3O
4/ HPEI compares, because the modification of the PEG of particle periphery causes Fe
3O
4/ HPEI.PEG size increases slightly, and can find out that the macromole layer of particle periphery thickens by naked eyes from TEM figure, has therefore proved that further PEG successfully modifies particle surface (with reference to the accompanying drawings 6b and table 2); The Zeta test result shows, after the PEGization reaction, shows No. 3 Fe
3O
4/ HPEI.PEG is positively charged (30.4mV) still, with Fe
3O
4/ HPEI compares, and electromotive force descends to some extent, and the ydrodynamics diameter is about 133.93nm, and dispersion of materials is more stable.These results show that PEG is modified at Fe equably
3O
4On the nano grain surface.
The synthetic Fe of table 2PEGization reaction
3O
4/ HPEI.PEG
Annotate: a is TEM result.
Embodiment 7
Get No. 1 Fe of above-mentioned preparation
3O
4/ HPEI aqueous solution 2mL adds 80 μ L triethylamines (density is 0.726 ~ 0.729g/mL, and concentration is 99.0%).Vibrate after 30 minutes abundant mixing, dropwise add 60 μ L diacetyl oxides (density is 1.08g/mL, and concentration is 98.5%) (triethylamine, diacetyl oxide and Fe
3O
4Among/the HPEI-NH
2Mol ratio=5:5:1), vial is placed on the TS-2 type shaking table (Jintan City positron in morning instrument plant), shaking table reaction 24 ~ 32 hours makes acetylizad No. 1 Fe in surface
3O
4/ HPEI.Ac nano particle.
By No. 1 Fe of Zeta electric potential testing method monitoring material
3O
4The acetylization reaction of/HPEI.Ac; potential drop is to being about 26mV after the acetylization reaction; electromotive force has obvious decline; but also not reaching electric neutrality, may be because the HPEI part is amino and ferric oxide interacts, so that their reactive behavior weakens; fully acetylize; so also present a part of positive polarity, but because they and ferric oxide effect, so can't be as simple HPEI to the toxicity of cell.With reference to subordinate list 3;
Get No. 1 Fe of above-mentioned preparation
3O
4/ HPEI 2mL DMSO solution separates by magnetic, removes upper solution, adds 2mLDMSO, by ultrasonic nano particle is dispersed among the DMSO, gets the 27.7mg succinyl oxide and is dissolved among the 4mL DMSO (dimethyl sulfoxide (DMSO)), to Fe is housed
3O
4Dropwise add the DMSO solution that contains succinyl oxide in the vial of/HPEI, wherein, succinyl oxide and Fe
3O
4The mol ratio of/HPEI amino is 5:1, shaking table reaction 24 ~ 32 hours, and centrifuge washing makes the surface and is the Fe of negative charge
3O
4/ HPEI.SAH nano particle.
By No. 1 Fe of Zeta electric potential testing method monitoring material
3O
4The carboxylation reaction of/HPEI.SAH, the particle surface electromotive force is negative electricity, No. 1 Fe after the reaction
3O
4/ HPEI electromotive force is reduced to-17.3mV from 38.07mV, and the result shows, the carboxylation reaction success, and amino fully reacts.With reference to subordinate list 3;
Table 31 Fe
3O
4The surface potential of-HPEI and finishing after product and ydrodynamics diameter (mean value ± standard deviation)
Embodiment 9
No. 1 Fe with above-mentioned preparation
3O
4/ HPEI, No. 1 Fe
3O
4/ HPEI.PEG, No. 3 Fe
3O
4/ HPEI and No. 3 Fe
3O
4/ HPEI.PEG material records the content of Fe element in the solution by the ICP-AES method of testing, be followed successively by 0.002,0.005,0.01 and the aqueous solution 2mL of 0.02mM/L with EP pipe preparation Fe concentration again, passes through T
2The T of MRI investigation material
2Relaxation effect.The result shows that relaxation rate increase along with concentration of iron in concentration of iron 0-0.05mM concentration range of these 4 kinds of materials has good linearly dependent coefficient.No. 1 Fe
3O
4/ HPEI(137.11mM
-1s
-1), No. 1 Fe
3O
4/ HPEI.PEG(157.61mM
-1s
-1), No. 3 Fe
3O
4/ HPEI(156.23mM
-1s
-1) and No. 3 Fe
3O
4/ HPEI.PEG(139.54mM
-1s
-1) four kinds of materials all have good T
2Relaxation effect and r
2Relaxation rate.And Fe
3O
4Even/HPEI material is modified macromolecule layer PEG, do not stopped itself and the contact rate of proton yet, on not too large impact of relaxation rate, and all have good relaxation rate (130-160mM
-1s
-1).Therefore, the Fe that the present invention synthesized
3O
4/ HPEI and Fe
3O
4/ HPEI.PEG nano material can be used as good T
2The signal attenuation contrast medium is expected to be applied to MRI molecular imaging diagnosis, and with reference to the accompanying drawings 7.
Can be better be used for bio-imaging in the body for the ease of material, this test determination No. 1 Fe
3O
4/ HPEI material through different modifying with the blood compatibility behind the different electric charges.Get human blood, at first centrifugal (2000rpm/min, 5min) and with PBS washing 5 times collects healthy red corpuscle.Again with No. 1 Fe
3O
4/ HPEI, Fe
3O
4/ HPEI.PEG, Fe
3O
4/ HPEI.Ac and Fe
3O
4/ HPEI.SAH material (50-400 μ g/mL) mix with red corpuscle leave standstill 2 hours after, behind the centrifugal 1min of 10000rpm/min, take pictures and survey the ultraviolet absorptivity of supernatant liquor.
Shown Fe among Fig. 8 No. 1
3O
4/ HPEI(Fig. 8 a), Fe
3O
4/ HPEI.PEG(Fig. 8 b), Fe
3O
4/ HPEI.Ac(Fig. 8 c) and Fe
3O
4/ HPEI.SAH(Fig. 8 d) the haemolysis result under different concns 50,100,200,400 μ g/mL.The absorbancy measurement of supernatant liquid is come the haemolysis effect of quantitative evaluation sample.Show such as Fig. 8 upper right corner uv-spectrogram, reaching under the 400 μ g/mL conditions Fe
3O
4/ HPEI, Fe
3O
4/ HPEI.PEG and Fe
3O
4The percentage of hemolysis of/HPEI.Ac is all less than 5%, but when material concentration reaches 400 μ g/mL, Fe
3O
4The hemolysis rate of/HPEI.SAH is 7.65%.This shows that they have good blood compatibility in given concentration range, thereby can make them be successfully used to biological in-vivo imaging; With reference to the accompanying drawings 8.
Embodiment 11
Take the KB cell as model cell, by vigor detection institute's synthetic materials of MTT colorimetric method for determining KB cell (a kind of cell strain of human epithelium's cancer) and the cytotoxicity after the process difference in functionality modification of surface.The KB cell respectively with No. 1 Fe
3O
4/ HPEI, Fe
3O
4/ HPEI.PEG, Fe
3O
4/ HPEI.Ac and Fe
3O
4Four kinds of materials of/HPEI.SAH were cultivated 24 hours under concentration is 10,25,50 and 100 μ g/mL and 37 ℃ altogether.Then, after processing, MTT measures light absorption value at 570nm place, and according to propagation and the vigor of this value calculating cell.The impact of material on cell proliferation and control group under the different concns (PBS damping fluid, pH 7.4) carry out statistical, implement by the one-way analysis of variance method.Compare with control group, the surface is through the Fe of modification and functionalization
3O
4/ HPEI.PEG, Fe
3O
4/ HPEI.Ac and Fe
3O
4/ HPEI.SAH in test concentration of iron 0 to 100 μ g/mL scope to the survival rate of KB cell showing property difference not.And Fe
3O
4/ HPEI begins to occur cytotoxicity (p＜0.05) when dosage reaches 50 μ g/mL, this shows it is unfavorable for cell when high density propagation, may be that toxic and surperficial high positive charge cell growth has certain influence to cell owing to surperficial HPEI.And the surface is through the Fe of modification and functionalization
3O
4/ HPEI.PEG, Fe
3O
4/ HPEI.Ac and Fe
3O
4/ HPEI.SAH then has good biocompatibility, can be used for medical use, and with reference to the accompanying drawings 9.
Comparative Examples
(people such as Ge, J.Phys.Chem.C 2009,113,13593-13599) with 1.25g FeCl according to document
24H
2After O and the 7.5mL ultrapure water supersound process, add again 6.5mL NH
3H
2O stirs 10min in air, reacted 3 hours under 134 ℃ of conditions in autoclave, washs with magnetic and separates, and obtains the Fe of 18.5nm diameter
3O
4Magnetic nanoparticle;
Claims (10)
1. the preparation method of the iron oxide magnetic nano particle of HPEI parcel comprises:
(1) the Fe source is added in the ultrapure water, add again NH
3H
2O also stirred in air, adds the hyperbranched polyethyleneimine HPEI aqueous solution of 0.02-0.12g/mL again, in 134 ~ 140 ℃ of reactions 3 hours; Reaction naturally cools to room temperature after finishing, and after washing of precipitate magnetic is separated, namely gets the ferroferric oxide nano granules Fe of HPEI parcel
3O
4/ HPEI;
(2) Fe
3O
4/ HPEI and methoxy poly (ethylene glycol) carboxylic acid carry out the PEGization reaction: with methoxy poly (ethylene glycol) carboxylic acid and 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride is dissolved in respectively among the 3-6mL DMSO, behind the stir-activating 3h, add again the Fe of step (1) preparation
3O
4/ HPEI, the hydromagnetic separating, washing is used in vibration again, among redispersion entry or the PBS, obtains the Fe of PEGization
3O
4/ HPEI.PEG nano particle;
Or Fe
3O
4/ HPEI and diacetyl oxide carry out acetylization reaction: with the Fe of step (1) preparation
3O
4/ HPEI nano particle is dispersed in the water, after triethylamine fully mixes, dropwise adds diacetyl oxide, and stirring reaction 24 ~ 48 hours is used the hydromagnetic separating, washing, makes acetylizad Fe
3O
4/ HPEI.Ac nano particle;
Or Fe
3O
4/ HPEI and butanedioic anhydride carry out carboxylation reaction: with the Fe of step (1) preparation
3O
4/ HPEI nano particle is dispersed among the DMSO, dropwise adds the DMSO solution that contains succinyl oxide, and stirring reaction 24 ~ 48 hours is used the hydromagnetic separating, washing, makes the Fe that carboxylated surface is negative charge
3O
4/ HPEI.SAH nano particle.
2. the preparation method of the iron oxide magnetic nano particle of a kind of HPEI parcel according to claim 1, it is characterized in that: the Fe source in the described step (1) is FeCl
24H
2O.
3. the preparation method of the iron oxide magnetic nano particle of a kind of HPEI parcel according to claim 1 is characterized in that: Fe source, ultrapure water, NH in the described step (1)
3H
2The proportioning of O is 1g:6-51mL:5mL.
4. the preparation method of the iron oxide magnetic nano particle of a kind of HPEI parcel according to claim 1, it is characterized in that: the Fe source in the described step (1) and hyperbranched polyethyleneimine HPEI mass ratio are 1-5:1.
5. the preparation method of the iron oxide magnetic nano particle of a kind of HPEI parcel according to claim 1 is characterized in that: the NH in the described step (1)
3H
2The O mass percent concentration is 25-28%.
6. the preparation method of the iron oxide magnetic nano particle of a kind of HPEI parcel according to claim 1 is characterized in that: the molecular weight Mw=25000 of the hyperbranched polyethyleneimine HPEI in the described step (1).
7. the preparation method of the iron oxide magnetic nano particle of a kind of HPEI parcel according to claim 1, it is characterized in that: the molecular weight of the methoxy poly (ethylene glycol) carboxylic acid in the described step (2) is 2000.
8. the preparation method of the iron oxide magnetic nano particle of a kind of HPEI parcel according to claim 1, it is characterized in that: the methoxy poly (ethylene glycol) carboxylic acid in the described step (2) is 1:5-10 with the mol ratio that is wrapped in the amino on ferroferric oxide magnetic nanoparticle surface.
9. the preparation method of the iron oxide magnetic nano particle of a kind of HPEI parcel according to claim 1 is characterized in that: the diacetyl oxide in the described step (2) or succinyl oxide and Fe
3O
4Among/the PEI-mol ratio of NH2 is 5-10:1.
10. the preparation method of the iron oxide magnetic nano particle of a kind of HPEI parcel according to claim 1 is characterized in that: triethylamine, diacetyl oxide and Fe in the described step (2)
3O
4Among/the PEI-the NH2 mol ratio is 5:5:1.
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